Ligand binding assays on microarrays in closed multiwell plates

ABSTRACT

Multiwell plates commonly used for immunoassay are increased in capacity and adapted for ease and speed of testing by forming a plurality of solid posts in each well of a plate. The posts and plate material and the dimensions of the posts are chosen to allow the immobilization of ligand patterns on an exterior wall of a post in a well and to permit a collimated beam of light directed to the post in a direction to achieve total internal reflection from a wall to generate an evanescent field in the plane of the ligands immobilized on the exterior wall of the post. The reflected light carries an image of localized intensity variations due to binding events between the ligand patterns and analytes in a sample introduced into a well. A cover plate seals the wells and provides for through holes for introducing sample material to the wells.

FIELD OF THE INVENTION

This invention relates to characterizing molecular binding events forperforming ligand binding assays and more particularly to systemsemploying ligand spots or microarrays in a closed multiwell format.

BACKGROUND

U.S. Pat. No. 6,594,011 issued Jul. 15, 2003, the entirety of which isincorporated by reference herein for all purposes, discloses an imagingapparatus and method for real time imaging ellipsometry for highthroughput sensing of binding events useful in molecular interactionanalysis including biotech applications. The apparatus disclosed employsthe immobilization of an array of binding or capture molecules(“ligands”) on a planar surface of a transparent substrate and the useof a beam of polarized light directed at the underside of the surface ina manner to achieve total internal reflection (TIR) and generate anevanescent field in the plane of the ligands. The ligands are exposed toa biological sample and analytes in the biological sample bind todifferent patterns of the immobilized ligands in a manner to change thepolarization at locations in the array at which binding occurs. An imageof the array is compared with a stored image of the initial lightpolarization shifts to determine the location and magnitude of bindingevents within the array, thus identifying and quantitating the analytespresent in the biological sample.

The apparatus for implementing the foregoing technique typically employsa prism or gratings to achieve the requisite TIR generated evanescentfield, the prism being the most practical implementation.

TIR imaging ellipsometry works well for fields of view up to 1-2 cm²,which permits real time imaging of tens of thousands of binding eventssimultaneously. However, there is a need to be able to image or scanareas which are much larger, such as 128 mm×86 mm (e.g. the area of a384 well or a 96 well multiwell plate), to permit lower costs per testand for multiple tests per patient for large numbers of patientssimultaneously, which is increasingly a requirement for clinicaldiagnostics and personalized medicine. Obviating the need for a prismsimplifies both the instrument and disposable multiwell plate.

SUMMARY

The present invention provides for total internal reflectionellipsometry without the use of prisms or gratings in a multiwell plate,thereby allowing for cost effective solutions for scaling themeasurement in a way previously unimagined. If the dimensions of postsare chosen so that a beam of light can be introduced through the postsin a manner to achieve total internal reflection from the side walls ofthe posts, ligands immobilized on the exterior face of each post will bein an evanescent field which enables binding events between ligands andanalytes in a biological sample in the well to be imaged simultaneously.In one application, the plurality of ligands form an array within eachwell where there are multiple posts per ligand spot. However, for largerconfigurations, it is possible to have arrays of ligands printed on theside walls of each post. The resulting image data is compared to astored image of the polarization phase distributions in the beamobtained prior to the exposure of the ligand array to a sample in orderto analyze the binding from the analytes in the samples.

The provision of solid transparent posts in the wells of a multiwellplate not only permits simultaneous imaging of binding events withoutthe necessity of a tag (e.g., fluorescent, chemiluminescent, radioactiveor otherwise) but also enables the placement of an array of separateligands for each well of a plate. This configuration allows forsimultaneous multiplexed assays for a number of patients for clinicaldiagnostics applications in one example. For rectangular posts with fourwalls, one embodiment includes the immobilization of a single ligand toeach wall making four ligands per post. With, for example, 1000 posts inevery well, for a ninety-six well plate, 384,000 individualligand/analyte interactions can be measured in a single disposable. Thisrepresents a substantial increase in the number of possible multiplexedassays provided along with a significant reduction in the cost perassay. The formation of ligand arrays on upright walls of posts and theuse of imaging ellipsometry to image binding events at the arrays thusrepresents a significant advance in the art.

The present invention also reduces a characteristic problem inevanescent field detection technologies, which is the problem ofsediment from a sample falling down onto the detection area duringmeasurement. Applications involving open well plates require that theplates be oriented with the open end upward. Having ligands at thebottom of open wells oriented in this manner exacerbates the sedimentproblem but this problem can be overcome with an array of ligands on theside wall as disclosed in the present invention.

In one embodiment, a transparent disposable multiwell plate has apattern of wells, identical to the pattern commonly used in commercialmultiwell plates. But in accordance with the present invention, eachwell includes a plurality of solid transparent posts each of a heightequal to the height of the wells in one example. The wells and posts maybe formed as a single part out of glass or plastic such that the heightof the posts and the wells defines the thickness of the sample regionwhich is enclosed after the bonding with a second plate. An array ofligand patterns is formed on the planar walls of the posts so that thereflected light from each post provides information regarding theinteraction of analytes in solution to a plurality of different ligands.

In a second embodiment, microarrays are formed on the exterior surfacesof the posts, conveniently by printing, as shown in FIG. 1 where thehalf circles represent the ligand spots immediately after printing andbefore drying or washing. A second transparent plate is bonded to thefirst plate to seal the wells. In one example, the second plate has atleast a pair of through holes aligned with each well in the first plate.The arrangement of holes allows for both the introduction of a sampleinto a well and the escape of excess sample material from the same well.

The lateral dimensions (x and y) and the height of each post herein arechosen to allow a beam of light to be directed to a post (e.g., into thebottom of each post) in a manner to achieve total internal reflection(TIR) from the (interior) face of at least one side wall of a post andto produce an evanescent field in the plane of the immobilized ligandson the exterior face of that wall.

The microarrays conveniently comprise different ligands immobilized inrows and columns such that the individual ligands are associated withposition information useful to identify the positions in the array atwhich binding events occur. The invention herein takes advantage of sucharrays which can be printed within each well.

Ligands on a selected post may be accessed by a collimated, polarizedbeam individually, or all the like-facing arrays on all posts in, forexample, rows of wells may be accessed simultaneously. Also, ligands onposts may be accessed sequentially by reorienting the disposable withrespect to the light source and imaging system (or vice versa) or byscanning individual spots using a laser.

Also, in one embodiment, the posts are coated with a metallic film andthe angle of the light source is varied to produce surface plasmonresonance at the SPR angle.

The scope of the invention is defined by the claims, which areincorporated into this section by reference. A more completeunderstanding of embodiments of the present invention will be affordedto those skilled in the art, as well as a realization of additionaladvantages thereof, by a consideration of the following detaileddescription of one or more embodiments. Reference will be made to theappended sheets of drawings that will first be described briefly.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective top view of a portion of a multiwell disposable;

FIG. 2 is a sectional view of a portion of the multiwell disposableillustrated in FIG. 1 in accordance with the principles of thisinvention;

FIG. 3 is a perspective view of a cover plate to be juxtaposed with theportion of FIG. 1 to form a sealed disposable;

FIG. 4 is a schematic side view of two wells of a sealed disposable withthe portions of FIG. 1 and FIG. 3 in mated positions;

FIG. 5 is a top view of a portion of a multiwell disposable inaccordance with another embodiment of the present invention;

FIG. 6 is a perspective view of a single well of the multiwelldisposable portion of FIG. 5 including a plurality of posts inaccordance with this invention;

FIG. 7 is a perspective view of a cover plate for the embodimentillustrated in FIGS. 5 and 6;

FIG. 8 is a schematic side view of two wells of a sealed disposable withthe portions of FIG. 5 and FIG. 7 in mated positions;

FIG. 9 is a schematic representation of the interrogation and imagingsystem for sensing binding events with the disposable of FIGS. 1, 2, 3and 4 or of FIGS. 5, 6, 7 and 8.

Embodiments of the present invention and their advantages are bestunderstood by referring to the detailed description that follows. Itshould be appreciated that like reference numerals are used to identifylike elements illustrated in one or more of the figures. It should alsobe appreciated that the figures may not be necessarily drawn to scale.

DETAILED DESCRIPTION

The present invention provides an advantageous apparatus and method forperforming ligand binding assays using microarrays in a multiwell plateformat. Prior to describing embodiments of the present invention indetail, the following definitions are provided for use throughout thepresent document.

DEFINITIONS

Microwell Plate: A flat plate with multiple “wells” used as small testtubes. The microplate has become a standard tool in analytical researchand clinical diagnostic testing laboratories with 6, 24, 96, 384 or even1536 sample wells arranged in a 2:3 rectangular matrix in one example.

Ligand: Any molecule that binds to another, in normal usage a solublemolecule such as a hormone or biological molecule that binds to abinding partner or capture molecule. The decision as to which is theligand and which is the capture molecule is often arbitrary. In thesense of this invention, the ligand refers to that binding elementattached to a planar surface and which binds to an analyte molecule in abiological sample.

Total Internal Reflection (TIR): An optical phenomenon that occurs whenlight strikes a medium boundary at a steep angle. If the refractiveindex is lower on the other side of the boundary (i.e., the side thatdoesn't directly receive the light) no light can pass through andeffectively all of the light is reflected. The critical angle is theangle of incidence where total internal reflection begins and continuesup to angles of incidence of 90 degrees.

Ellipsometry: A very sensitive optical measurement technique providingunequalled capabilities for thin film analysis utilizing the change ofpolarization of light which is reflected off a sample or transmittedthrough a sample.

Surface Plasmon Resonance (SPR): The excitation of surface plasmons bylight is denoted as a surface plasmon resonance for planar surfaces.Plasmons are collective oscillations of large numbers of electrons inmatter, mostly in metals.

Arrays: Ligands affixed to a surface at separate localized regionscalled spots in an ordered manner thus forming a microscopic patternwhere ligand identity is determined by the location (or “address”) ofthat particular spot.

Binding Protein (Ligand) Assay: A test that uses the binding of proteins(e.g., antibodies) to other ligands (e.g., antigens) to identify andmeasure the concentration of certain biological substances in blood,urine or other body components. Ligand assays may be used to diagnosedisease, drug or vitamin levels, response to therapy or otherinformation of biological relevance. Also, test results can provideinformation about a disease that may help in planning treatment (forexample, when estrogen receptors are measured in breast cancer).

FIG. 1 is a perspective top view of a transparent plate 10 that is aportion of a multiwell disposable, and FIG. 2 is a sectional view ofplate 10 along line A-A illustrated in FIG. 1 in accordance with theprinciples of this invention. Plate 10 has an array of wells 12 formedtherein and each well includes a plurality of solid posts 13. In oneexample, there may be 49 posts in each well. In one embodiment, plate 10will have 96 or 384 wells and have the overall dimensions of 12.8 cm by8.6 cm with a plate thickness of roughly 1 mm so that the lateraldimensions are in conformance with dimensions of available multiwelldisposables.

For a 96-well plate as an example, a well has dimensions of 7 mm by 7 mmfor square wells or a diameter between 6 and 7 mm for circular wells,and each post is solid and has a height between about 25 μm and about100 μm to be substantially equal to the depth of the shallow wells. Thefour well section of plate 10 which is shown in FIG. 1 illustrativelyhas dimensions of 9 mm by 9 mm in the 384-well example and 18 mm by 18mm in the 96-well example.

FIG. 3 shows a plate 20 which has dimensions equal to those of plate 10.Plate 20 is juxtaposed with and bonded to plate 10 in a manner tosubstantially cover or seal wells 12 (see, e.g., FIG. 4). The two plates10 and 20 have the same index of refraction and are bonded together withknown techniques to avoid optical reflection at the interface. In thisparticular design, plate 20 includes a pair of through holes 21 and 22,with each pair in registry with a well. Illustrative pairs of holes areshown in FIG. 3 and correspond to each of the four wells of FIG. 1. Thethrough holes provide access for the introduction of a sample into awell and for excess sample material to escape the well. Various designsmay be used for plate 20 to allow a continuous flow of liquid or gasinto one hole while the gas or liquid inside may be exchanged.

FIG. 4 shows a schematic side view of two wells 12 with plates 10 and 20joined together. It should be understood that the wells and the postsare of like height and so are optically continuous with plate 20 in oneembodiment. The purpose of this is to allow any light which reflectsfrom a post side wall to be transmitted through plate 20 and out of theplate for detection.

FIG. 5 is a top view of a transparent plate 30 that is a portion of amultiwell disposable in accordance with another embodiment of thepresent invention, and FIG. 6 is a perspective view of a single well 32of the multiwell disposable portion of FIG. 5. As shown in FIGS. 5 and6, wells 32 have a square well design in accordance with thisembodiment. Wells 32 are formed in plate 30 and can be seen to include aplurality of posts 33 shown, illustratively, in an array pattern. In oneexample, each well includes a 13×13 array of posts 33 and has dimensionsof 7 mm×7 mm in this particular design with each post having dimensionsto permit TIR. as described hereinbefore. A space 35 is provided,conveniently, in the center of the array to align with the hole of acover plate (see, e.g., FIG. 7) which will act as the guide for sampleentry.

The embodiment as shown in FIGS. 5 and 6 can be adapted to a multiwellplate in which each well has a relatively large number of posts. It ispossible in one embodiment for the ligand spots to be smaller than thedimensions of the posts. In this embodiment, one may print ligand arrayson the side walls of the posts. Since typical ligand spots are on theorder of 100 microns, this would be more practical with posts having atleast one dimension greater than three times the diameter of a spot orgreater than 300 microns in this case. For example, for 2D arrays onsidewalls, posts could be 1 cubic millimeter with each side wallcontaining 25 ligand spots. For 1D arrays, the posts can be made 100microns high, 100 microns thick, and 1 mm wide to accommodate five spotson the longer side wall. Minimizing the height of the posts isadvantageous for allowing one to use very small sample volumes.

In another embodiment of the type shown in FIGS. 5 and 6, the postdimensions are smaller than the dimensions of microarray spots. Ligandspots may be introduced to subsets of the posts in a well where theycompletely cover multiple posts (e.g., see spots 37 in FIGS. 2 and 5).The surface of plate 30 is treated with a surface chemistry which bindstarget ligands. The ligand spots dry on the posts leaving the post topand sidewalls coated with a single ligand. There are still multipleligand arrays per well since each spot 37 may be a different ligand butnot per post as in the first embodied description. Accordingly, eachspot 37 occupies a known position. In this illustration, sixteen ligandspots are shown for each well and, thus, up to sixteen individual testscan be carried out for each sample introduced into a well in thisparticular design. Any binding which occurs between an analyte in thesample and the ligands of a spot will occur at a known (spot) position.

After the ligand spots dry, plate 30 is rinsed thoroughly to removeexcess material and a cover plate 40 of FIG. 7 is bonded to plate 30 toseal the wells. In this particular design, plate 40 includes a centerhole 42 in registry with each square well and four exit slits 44 a, 44b, 44 c, and 44 d per well to allow uniform flow of fluid radially fromthe square well centers. Through holes 42 are aligned with space 35(FIGS. 5 and 6) for the introduction of a sample to the well. Plate 40would have dimensions equal to plate 30 (FIG. 5) for this design and bebonded in a manner substantially similar to what was describedpreviously. The inside border of the four slits 44 a-44 d is interior tothe outer border of each well 32 in FIG. 5 so that air and fluid areallowed to escape during filling. Advantageously, FIG. 7 illustrates acover plate 40 with a practical arrangement for fluid entry and exitapertures for a four well embodiment.

It is to be understood that the use of posts in a well, in accordancewith the principles of this invention is practical whether a pluralityof like ligands or an array of ligand patterns are immobilized on postsidewalls (and tops) or whether the posts are smaller or larger than thedimensions of the ligand spots.

FIG. 8 shows a schematic side view of two wells 32 with plates 30 and 40joined together and hole 42 aligned with space 35 and slits 44 a and 44c properly aligned.

FIG. 9 shows illustrative posts 73 with arrays 71 of ligands immobilizedon the exterior of two walls of each post. It should be understood thatother walls may also be utilized. It is also to be understood that thepatterns of ligands are immobilized in an array in which the position ofa ligand(s) in the array is significant. The reason for this is that abinding event between a ligand and an analyte in a sample in a wellcauses a change in the relative phase between s- and p-polarizedreflected light at that position. In accordance with this invention animage of all those phase changes is compared with a stored initializedimage to determine which analytes are present in a sample.

FIG. 9 shows a light source 102 and an imaging system 104 operative todirect a beam of polarized light at a wall of a post 73 in a manner togenerate an evanescent field in the plane of the ligands. Analytes in asample introduced into a well through, for example, an aperture 75 willbind with different patterns of ligands in the array providing forlocalized intensity changes in the reflected light at the positions ofthe ligands effected. Examples of applicable light sources and imagingsystems are disclosed in the above-noted U.S. Pat. No. 6,594,011, whichhas been previously incorporated by reference herein.

In one example, in order to image binding events, the ligand array isaccessed by collimated, polarized light directed from below plate 70 ata post wall in a manner to obtain TIR at the wall. This is to becontrasted with previous arrangements typical in the art operative toachieve TIR at the horizontal bottom surface of the plane on whichligand arrays are immobilized.

In order to achieve total internal reflection (TIR) for imaging thepattern of binding events on a wall of a post in the array of wells ofFIG. 9, the indices of refraction of the materials as well as the anglebetween the transmission surface and the TIR surface is importantbecause TIR cannot occur unless the following equation is satisfied:sin(A)*√{square root over (n ₂ ² −n ₁ ² sin²(B))}−cos(A)*n ₁ sin(B)≧n ₃where A is the angle between the transmission surface contacting air andthe TIR surface contacting the sample, B is the incidence angle of thelight in air, n₁ is the index of refraction of air, n₂ is the index ofrefraction of the transparent material of the plate and n₃ is the indexof refraction of the sample (water, blood, urine, etc.). This formulasimplifies dramatically if the wall of the well is normal to the bottomof the plate since then A=90° and the second term in the above equationvanishes. The resulting equation becomes:

${\sin(B)} \leq {\frac{1}{n_{1}} \times \sqrt{n_{2}^{2} - n_{3}^{2}}}$

In one embodiment compatible with current multiwell plate technology,individual wells of the plate are spaced according to Ansi/SBS standards(e.g., 4.5 mm center to center for 384 well plates and 9 mm center tocenter for 96 well plates). The spacing of the wells is true tostandard. But, in accordance with the principles of this invention, theaddition of posts to a well and the dimensions of individual posts areimportant. In fact, there is a minimum post thickness in at least onedimension to allow total internal reflection to occur at precisely thecritical angle from the side wall in such a way that the entire surfacemay be illuminated. This minimum post thickness depends on the index ofrefraction of the plate, the index of refraction of the material insidethe wells during a measurement, and the height of the posts. The formulafor post thickness in the direction of light propagation is thefollowing:

$t \geq \frac{h\sqrt{n_{2}^{2} - n_{3}^{2}}}{n_{3}}$where t is the post thickness, h is the post height, n₂ is the index ofrefraction of the plate and n₃ is the index of refraction of the(sample) material inside the well during measurement. All variables inthe equation are labeled in FIG. 9. For example, for plastic plateshaving a refractive index of 1.49, a post height of 75 microns, and aliquid sample of index 1.34, the minimum thickness is 36.5 microns. Forrefractive indices of acrylic plates and typical biological fluidsamples, the height of the posts cannot be much more than double thepost thickness. For simplicity, all of the Figures including postswithin sample wells are drawn with t=h since this is a convenientgeometry for purposes of illustrating certain embodiments. Additionally,TIR would work for any index below 1.34 for the sample material sincethe same angle would be in the TIR region. For polycarbonate plates ofindex 1.59 and the same liquid sample, the minimum thickness of a 75micron high post would be 47.9 microns if imaging is needed all the wayto the TIR angle in such a way that the entire side wall is illuminated.

The manner in which the arrays are printed is well understood anddescribed in the literature on topics dealing with technological aspectsof DNA and protein microarrays. Contact and non-contact printingtechniques for ligands are applicable. Ligands may be immobilized on allthe (exterior) walls of a post or several posts with a small drop ofsolution larger than the dimensions of the individual posts. However,when plates 10 and 30 are joined, the array on the top of a post becomesinaccessible and does not affect signals generated in the lightreflected from a side wall since the refractive index of the protein isclose to the curing adhesive which binds the two parts together.Furthermore, optical thin films may be provided on the sidewalls toamplify signals within the evanescent field region on those surfaces.

The interrogating beam from source 102 may be directed at the (ligand)wall of a single post or simultaneously at the (ligand) walls of aplurality of posts which are in the cross section of the collimatedbeam. Moreover, the beam may be redirected by conventionalimplementations (i.e., small scanning mirrors) to access all the wellsof each of a sequence of rows and columns of FIG. 1 or 6.

In accordance with another embodiment of the present invention, theapparatus of FIGS. 1-9 can be used also in conjunction with an opticalsystem employing surface plasmon resonance (SPR) to sense the bindingevents between analytes in a sample and the ligands of an array. In thisembodiment, a metallic film (not shown) of gold, silver, or copper,typically ˜50 nm thick in the case of gold, is formed on the sidewallfaces of posts and ligand arrays are immobilized afterward within eachwell. The interrogating light beam is directed at a post wall at anangle within the TIR regime in a manner to produce surface plasmonswithin the metal film with a characteristic evanescent field whichinteracts with the plane of the array. In this embodiment, the beamangle is no longer the critical angle but the SPR angle within the TIRregime as is well understood in the field.

The disposable multiwell plate in accordance with the present inventionhas a number of differences and benefits over prior uses of imagingellipsometry.

-   -   1. It is clear that the apparatus and method herein disclosed        comprises a disposable multiwell plate which has dimensions        compatible with robotic instrumentation currently in use with        multiwell plates.    -   2. The disposable multiwell plate is employed similar to        commercially available multiwell plates in terms of manual or        automatic fluid dispensing.    -   3. No prism or index matching oils are necessary for the user to        achieve total internal reflection.    -   4. Signal is far less sensitive to false readings by sediment        falling out of solution.    -   5. Completely separate assays can be achieved within the same        plate simply by rotating a multiple of 90 degrees or reading the        plate from different side walls of the posts.    -   6. Significantly more imaging or scanning area is now achievable        without the expense of the large optics (e.g., 8″) otherwise        required or an array of small prisms accurately aligned        underneath each well or row of wells.    -   7. Because the ligands are sealed inside a microfluidic        multiwell plate, the disposable does not need to be stored in a        clean air environment as is the case with open multiwell plates        with ligands on the bottom of wells which could collect dust.        For ellipsometry, surfaces need to be kept very clean and this        is one of the reasons why ellipsometry has not been used in        conventional diagnostics in the past.    -   8. For the microfluidic embodiment in particular, fluid volumes        are only 2.5 μl per well for 96-well plates and 450-600 nl per        well for 384-well plates. Typical volumes are around 100 μl per        sample and it is advantageous to use the least amount of sample        as possible for expensive samples.    -   9. The thin, planar design (−2-3 mm thick) of the microfluidic        embodiment allows numerous plates to be stacked for high        throughput applications. For example, 100 stacked plates is only        about 1 foot high and could include tests for 38,400 patients.

In the case of the imaging system 104, the camera itself need not be asophisticated, high resolution CCD camera if, indeed, relatively fewligands are immobilized on each ligand wall as would be the case formost current diagnostic use. In such cases on the order of a few to tensof ligands need be immobilized for diagnostic purposes. This lowerdemand for content translates to less expensive cameras and morecost-effective systems configured for doctor offices and clinicallaboratories performing diagnostic testing. For other purposes such asbiological research in metabolic or cancer pathways, drug targetinteractions in drug discovery, and biomarker profiling during clinicaldevelopment of new therapeutics, higher content of ligands in arrays(100's to 1000's) may be required.

What has been described is considered merely illustrative of theinvention herein and it is apparent that various modifications thereofmay be devised by those skilled in the art still without departing fromthe spirit and scope of the invention as encompassed by the followingclaims. For example, a post need not be rectangular in shape. It needonly have at least one planar wall on which ligands are immobilized. Ifmore than one wall of a post is planar, ligands can be immobilized oneach planar wall and the plate can be reoriented with respect to thelight source and imaging system (or vice versa in which the light sourceand imaging system can be reoriented with respect to the plate) toproduce the requisite TIR operation. Also, although simultaneous accessof multiple ligand arrays as well as scanning from one ligand array toanother have been described herein, it is contemplated that theindividual spots of an array can be scanned in the manner describedherein, for example, by a laser. Consistent with conventional multiwellplate usage, the wells may be left open or the well array may be sealedas described herein.

What is claimed is:
 1. An apparatus for forming ligand binding assaysfor a plurality of analytes in a sample, comprising: a first planartransparent plate including a first well therein; and a firsttransparent solid post within the first well, said post having a heightsubstantially equal to the height of said well and having at least oneplanar wall, wherein said post has a thickness$t \geq \frac{h\sqrt{n_{2}^{2} - n_{3}^{2}}}{n_{3}}$ where t is the postthickness along the direction of a light beam propagation, h is theheight of the post, n₂ is the index of refraction of the plate, and n₃is the index of refraction of a sample introduced to the well.
 2. Theapparatus as in claim 1, wherein said post has at least one planar sidewall having an array of ligands immobilized thereon.
 3. The apparatus asin claim 2, wherein said post is of a material and has cross-sectionaldimensions and a height to permit a beam of light directed at the postto produce total internal reflection at said at least one planar sidewall and to generate an evanescent field in the plane of the array ofligands immobilized thereon.
 4. The apparatus as in claim 3, furthercomprising a second transparent plate juxtaposed with said first platein a manner to seal said well, said second plate including a pair ofthrough holes which is aligned with said well.
 5. The apparatus as inclaim 3, wherein said post has a rectangular cross section with fourplanar walls.
 6. The apparatus as in claim 5, further comprising asource of a collimated beam of polarized light and means for directingsaid beam into the bottom of said post.
 7. The apparatus as in claim 6,further comprising means for capturing the image of the light intensitypattern reflected from the plane in said evanescent field.
 8. Theapparatus as in claim 2, wherein said side wall includes a metallic filmthereon and said plurality of ligands is formed on said film.
 9. Theapparatus as in claim 1, wherein said plate includes a plurality ofwells and each of said wells includes a plurality of posts, each posttherein having at least one planar wall having an array of ligandsimmobilized thereon.
 10. The apparatus as in claim 9, wherein each ofsaid posts is of a material and has cross sectional dimensions and aheight to permit a beam of light directed to said posts to produce totalinternal reflection at the at least one planar wall of each post and togenerate an evanescent field in the plane of the array of ligandsimmobilized on the at least one planar wall of each post.
 11. Theapparatus as in claim 10, further comprising a second transparent platejuxtaposed with said first plate in a manner to seal said wells, saidsecond plate including a plurality of through holes, each of saidplurality of through holes being aligned with at least one of said wellssample to a well.
 12. The apparatus as in claim 10, further comprising asource of a beam of polarized light and means for directing said beam atdifferent walls of said posts.
 13. The apparatus as in claim 12, furthercomprising means for capturing the image of the light intensity imagereflected from a wall.
 14. An imaging apparatus, comprising: a firstplanar transparent plate including a first well; and a first transparentpost within the first well, said post having at least one planar wall,wherein said post has a thickness$t \geq \frac{h\sqrt{n_{2}^{2} - n_{3}^{2}}}{n_{3}}$ where t is the postthickness along the direction of a light beam propagation, h is theheight of the post, n₂ is the index of refraction of the plate, and n₃is the index of refraction of a sample introduced to the well.
 15. Theapparatus as in claim 14, wherein said post has at least one planar sidewall having an array of ligands immobilized thereon.
 16. The apparatusas in claim 15, wherein said post is of a material and hascross-sectional dimensions and a height to permit a beam of lightdirected at the post to produce total internal reflection at said atleast one planar side wall and to generate an evanescent field in theplane of the array of ligands immobilized thereon.
 17. The apparatus asin claim 14, further comprising a second transparent plate juxtaposedwith said first plate in a manner to seal said well, said second plateincluding a pair of through holes which is aligned with said well. 18.The apparatus as in claim 14, further comprising means for capturing animage of the light intensity pattern reflected from the plane in saidevanescent field.
 19. The apparatus as in claim 14, further comprisingmeans for capturing an image of spatially distributed polarizationchanges in a light beam reflected from the plane in said evanescentfield.
 20. The apparatus as in claim 14, wherein said plate includes aplurality of wells and each of said wells includes a plurality of posts,each post therein having at least one planar wall having an array ofligands immobilized thereon.